Although several immunological reagents have been developed (ex: tumor infiltrating lymphocytes, antigen specific tumor vaccines etc.) during the last few years which can recognize and kill melanoma cells and have the potential of curing melanoma patients, most patients are resistant to this form of therapy. This resistance is probably dependent upon a complexity of variables affecting the balance between activation of the immune system and growth of tumor cells in vivo and our limited understanding of them. My work is focused on analyzing variables potentially affecting this balance. During the last year we have further addressed several issues related to this topic, specifically: 1. The quality and quantity of the immune response to peptide-based vaccination. Our data suggest that the precursor frequencies obtained using the current vaccination regimens are not comparable with the precursor frequency of T cell specific for exogenous pathogens during acute or chronic infections. Preliminary work suggests that different schedules of vaccine administration could enhance the immune responses against cancer.To reach this conclusion several technologies were implemented in our laboratory such as preparation and utilization of soluble HLA/epitope tetramers, TaqMan-based real time PCR, intra- cellular FACS technology and development of a technique allowing to expand tumor/T cell pairs from fine needle aspirates of metastatic melanoma. Addressing this issue several publications are presently in press.In line with previous work we have noted that although T cell responses can be elicited during vaccination with melanoma epitopes this localize at tumor site only when suggificient target antigen is present. We have documented during the last year that in several cases melanoma antigen expression rapidly descreases during vaccination treatments and this is associated with decreased localization of vaccine-specific T cells at tumor site.Last year work will be continued this year by trying to quantify more in detail the requirements for T cell activation necessary for tumor rejection. This will be done by analysis of T cell responses after various numnber of vaccinations and the development of a murine model for the analysis of the same question. - Human Subjects & Human Tissues, Fluids, Cells, etc.